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Protocol no. 47 
HET-CAM TEST 

The potential irritancy of compounds may be detected by observing adverse changes which occur in the chorionallantoic membrane of the egg after exposure to test chemicals. 

CONTACT

Dr med. H. Spielmann 
Direktor und Professor
ZEBET Bundesgesundheitsamt
Postfach 33 00 13 
D-W-1000 Berlin 33 Germany 
Tel: Germany - 30 8308 2247 Fax: Germany - 30 8308 2741 

RATIONALE 

Chemicals are placed directly onto the chorionallantoic membrane of the hen's egg. The occurrence of vascular injury or coagulation in response to a compound is the basis for employing this technique as an indication of the potential of a chemical to damage mucous membranes (in particular the eye) in vivo. 

BASIC PROCEDURE

Hen's eggs are rotated in an incubator for 9 days after which time any defective eggs are discarded. The shell around the air cell is removed and the inner membranes are extracted to reveal the chorionallantoic membrane. Test chemicals are added to the membrane and left in contact for 5 min. The membrane is examined for vascular damage and the time taken for injury to occur is recorded. Irritancy is scored according to the severity and speed at which damage occurs. video 

CRITICAL ASSESSMENT

The test has several advantages including its simplicity, rapidity, sensitivity, ease of performance and its relative cheapness. A factor to consider is the fertility and the ability of the eggs to hatch. The survival of chickens is dependent on a complex interrelationship of ecological factors (e.g. the genetic background and the age of the mated birds, the nutritional status and general management of the flock, and in part seasonal variations). Eggs should, therefore, be obtained from reliable local contractors. (The authors have produced some empirical data on the fertility of the particular flocks they use. The fertility of middle-aged flock is approximately 90% with 10-15% defective eggs. On average there are 20% lesions produced during preparation.) The major disadvantage of the procedure is the subjective nature of the evaluation of the results. This is overcome to a certain extent by the inclusion of positive standards and by using a comprehensive scheme for scoring the irritant effects of the chemicals. The exposure period of 5 minutes to the test chemical has been found to be sufficient to reveal irritant/toxic effects (longer exposure does not appear to yield any additional information). A factor for consideration is whether the Hen's egg test may be considered as an animal experiment. At present the test is often looked upon as being borderline, although it has potential to be used in a manner likely to reduce the number of mammals used in conventional testing and also to contribute towards a reduction in the associated suffering. 

TEST STATUS

This test, along with several other in vitro systems, is presently undergoing validation as an alternative test to replace the Draize Rabbit Eye Test, in a national interlaboratory study started in June 1988, by the Federal Health Office (BGA) of the Federal Republic of Germany (FRG). The aim of this collaborative study is to validate the classification of chemicals, with regard to their irritation potential, using the Neutral Red/Kenacid Blue (NR/KB) cytotoxicity assay and the Hen's Egg Test Chorioallantoic Membrane (HET-CAM) assay according to Lpke. The FRG Public Health Office (BGA) is coordinating the scheme which includes, 12 toxicology laboratories in the chemical industry, universities, the BGA and other research institutions who will study 44 substances with a variety of chemical, biochemical, and toxicological characteristics. The validation test is intended to provide comparative data for the development of an alternative routine test scheme, and which is performed under routine laboratory conditions. The validation project of alternatives for the Draize test consists of three parts: a preliminary phase, an interlaboratory assessment, and, finally, the development of a database of results. During the preliminary phase the cytotoxicity test and the HET-CAM assay have been established in the different laboratories. The participants have agreed on standard and mandatory protocols and on the choice of chemicals. Two preliminary trials have been performed with 4 test substances. During the interlaboratory assessment 35 chemical substances of a variety of chemical structure groups have been tested with both alternative techniques in 12 laboratories under conditions that will be defined in the preliminary phase of the study. The main purpose of the validation phase is the comparative and statistical evaluation of all data at the BGA followed by a final scientific validation which could prove of interest to regulatory authorities. This assessment determines both the reproducibility of the results within a given laboratory and of a given test between laboratories. Preliminary findings indicate that data from the HET-CAM test appears to correlate better than the two cytotoxicity tests when compared to in vivo Draize scores. The cytotoxicity tests give a greater number of false positives and negatives compared to the HET-CAM test. The cytotoxicity test have, however, given better reproducibility of test data, within and between laboratories, than the HET-CAM. This is most probably due to the automated determination of NR and KB values and to the highly subjective determination of the toxicological endpoints in the HET-CAM test which are difficult to standardize. In conclusion, both the cytotoxicity tests and the HET-CAM test can provide reproducible results if carried out under routine conditions with well trained operators. The third phase of the validation project, database development, commenced on June 1st, 1990. Seven laboratories are testing a total of 200 chemicals which again include a variety of chemical classes. 

CHEMICALS TESTED

Acetone Acetonitrile Acrylamide Aniline Ascorbic acid Benzalkonium chloride (0.5%) Benzoic acid 2-Butoxyethanol Copper(II) sulphate Cyclohexanol DEHP (100%) Dimethylsulphoxide EDTA-Na salt Ethanol Lactic acid (5%) n-Hexane Nicotinamide Nitrobenzene Phenol Propanediole 2-Propane-1-ol Pyridine SDS (1%) Sodium chloride Tetrachloroethane Thiourea Toluene 

REFERENCES

  1. Lopke, N.P. (1986) HET (Hen's Egg Test) in Toxicological Research. In: Skin Models. Models to Study Function and Disease of Skin (eds. Marks, R. & Plewig, G.). Published by Springer-Verlag, pp 282-291.
  2. Kalweit, S., Besolke, R., Gerner, I. & Spielmann, H. (1990) A national validation project of alternative methods for the Draize rabbit eye test. Toxic. in Vitro, 4, 702-706. 
  3. Spielmann, H., Gerner, I., Kalweit, S., Moog, R., Wirnsberger, T., Krauser, K., Kreiling, R., Kreuzer,H., Lupke, N.P., Miltenburger, H.G., Muller, N., Murmann, P., Pape, W., Siegmund, B., Spengler, J., Steiling, W. & Wiebel, F.J. (1991) Interlaboratory assessment of alternatives to the Draize eye irritation test in Germany. Toxic. in Vitro, 5 No.5/6, 539-542. IP-47 C January 1992 
IP-47 © January 1992